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The secretion of potential therapeutic factors by mesenchymal stem cells (MSCs) has aroused much interest given the benefits that it can bring in the field of regenerative medicine. Indeed, the in vitro multipotency of these cells and the secretive capacity of both angiogenic and immunomodulatory factors suggest a role in tissue repair and regeneration. However, during culture, MSCs rapidly lose the expression of key transcription factors associated with multipotency and self‐renewal, as well as the ability to produce functional paracrine factors. In our study, we show that a three‐dimensional (3D) culture method is effective to induce MSC spheroid formation, to maintain the multipotency and to improve the paracrine activity of a specific population of human amnion‐derived MSCs (hAMSCs). The regenerative potential of both 3D culture‐derived conditioned medium (3D CM) and their exosomes (EXO) was assessed …